By Richard Jefferys, Project Coordinator at Treatment Action Group (TAG)

In the spring of 2013, a wildly inaccurate Daily Telegraph story about HIV cure research claiming “there will be a breakthrough in finding a cure for HIV within months” was amplified around the Internet, understandably receiving a massive amount of attention and interest. To their credit, the researchers whose work prompted the piece published a statement addressing the inaccuracies, and eventually the Telegraph made extensive corrections. Recently, in the debut issue of The Lancet’s new online HIV journal, results from the trial at the center of the story were published - to no fanfare whatsoever.

The drug being tested was the HDAC inhibitor panobinostat, which has been reported to reverse HIV latency in laboratory experiments. In the 15-person phase I trial, a small but significant increase of approximately 2-3 fold in cell-associated HIV RNA was documented, and plasma viremia was more frequently detected compared to baseline. A transient decrease in the amount of total HIV DNA in the blood was captured at day 14 of the study, but otherwise no measures of the HIV reservoir (including levels of replication-competent virus) were altered by panobinostat. A subset of nine study participants subsequently underwent an analytical treatment interruption, and HIV viral load rebounded to over 1,000 copies in all cases within a median of 17 days (range 14–56). The main side effect of the drug was fatigue, and no adverse events of greater than grade 1 occurred. However, as noted in an accompanying commentary by Stephen Kent and Miles Davenport, HDAC inhibitors have been shown to cause long-term changes in expression of multiple genes, so long-term safety will need to be carefully monitored.

The panobinostat paper does offer a hint of a greater effect of the drug in four participants, but these types of post-hoc (not planned in the original study protocol) subset analyses have to be interpreted with great caution. In these individuals, a decline in total HIV DNA levels of around 67–84% is reported to have persisted, and was correlated with a slightly longer time to viral load rebound during the analytical treatment interruption. At the recent Strategies for an HIV Cure meeting, which took place at the National Institutes of Health in Bethesda in October, Mathias Lichterfeld presented some preliminary data associating this delayed time to viral load rebound with interferon-stimulated gene expression and natural killer cell activity, suggesting a role for innate immunity. Given the small number of participants and the fact that the analyses are all post-hoc, these results can only be considered exploratory and require confirmation.

Beyond the results obtained with individual candidates, there is a notable larger issue that looms over latency-reversing approaches generally. The experience of the Boston patients has demonstrated that significant HIV reservoir reductions of around 3 logs (1,000-fold) can lead to delayed viral load rebound after ART interruption, but not a cure. This is in line with mathematical modeling by Alison Hill (published recently in PNAS) which estimates that an HIV reservoir reduction more on the order of 5-6 logs (100,000-1 million-fold) would be needed to achieve a long-term cure in most people. In this context, the fact that no latency-reversing strategy has yet had any significant effect on HIV reservoir levels does not seem very encouraging. But, in case that sounds like doom saying, it’s also true that the work is at an early stage, and latency reversal is just one of many strategies being pursued in HIV cure research.

Background: Activating the expression of latent virus is an approach that might form part of an HIV cure. We assessed the ability of the histone deacetylase inhibitor panobinostat to disrupt HIV-1 latency and the safety of this strategy.

Methods: In this phase 1/2 clinical trial, we included aviraemic adults with HIV treated at Aarhus University Hospital, Denmark. Participants received oral panobinostat (20 mg) three times per week every other week for 8 weeks while maintaining combination antiretroviral therapy. The primary outcome was change from baseline of cell-associated unspliced HIV RNA. Secondary endpoints were safety, plasma HIV RNA, total and integrated HIV DNA, infectious units per million CD4 T cells, and time to viral rebound during an optional analytical treatment interruption of antiretroviral therapy. This trial is registered with ClinicalTrial.gov, number NCT01680094.

Findings: We enrolled 15 patients. The level of cell-associated unspliced HIV RNA increased significantly at all timepoints when patients were taking panobinostat (p<0·0001). The median maximum increase in cell-associated unspliced HIV RNA during panobinostat treatment was 3·5-fold (range 2·1—14·4). Panobinostat induced plasma viraemia with an odds ratio of 10·5 (95% CI 2·2—50·3; p=0·0002) compared with baseline. We recorded a transient decrease in total HIV DNA, but no cohort-wide reduction in total HIV DNA, integrated HIV DNA, or infectious units per million. Nine patients participated in the analytical treatment interruption, median time to viral rebound was 17 days (range 14—56). Panobinostat was well tolerated. 45 adverse events were reported, but only 16 (all grade 1) were presumed related to panobinostat.

Interpretation: Panobinostat effectively disrupts HIV latency in vivo and is a promising candidate for future combination clinical trials aimed at HIV eradication. However, panobinostat did not reduce the number of latently infected cells and this approach may need to be combined with others to significantly affect the latent HIV reservoir.

Purging latent HIV with reactivating drugs is one of the most promising approaches to curing HIV infection. In The Lancet HIV , Thomas Rasmussen and colleagues report results of a phase 1/2 trial 1 of panobinostat, a histone deacetylase inhibitor, to reactivate latent HIV. They show that the drug was well tolerated in the short term and the treatment increased both the amount of cell-associated HIV RNA and low-level transient HIV viraemia. There was, however, no overall change in measurements of the HIV reservoir after 8 weeks of panobinostat treatment.

Two recent papers address the potential of broadly neutralizing antibodies (bNAbs) to decrease HIV rebound from persistent reservoirs. In the journal Cell, Ariel Halper-Stromberg and colleagues report the results of experiments conducted in the humanized mouse model with a “tri-mix” of three monoclonal bNAbs: 3BNC117, 10-1074, and PG16. When administered as post-exposure prophylaxis four days after the mice were challenged with HIV, the bNAb tri-mix did not prevent infection, but there was a significant delay in viral load rebound after the treatment was stopped (compared to a control group of mice given combination antiretroviral therapy on the same schedule). The researchers conclude that the bNAbs were more effective than ART at preventing the formation of the latent HIV reservoir in the mice, likely due to antibody-mediated effector mechanisms facilitating the clearance of infected cells (similar to recent findings in the macaque model).

Subsequent experiments investigated the effects of combining the bNAb tri-mix with potential latency-reversing agents including vorinostat (an HDAC inhibitor), I-BET151 (a BET protein inhibitor) and CTLA (a T cell inhibitory pathway blocker). In humanized mice with established HIV infection, the combination of the bNAb tri-mix and all three latency-reversing agents significantly reduced the number of mice experiencing viral load rebound after therapy cessation compared to bNAbs alone (10 of 23 mice rebounded in the combination group versus 22 of 25 in the bNAbs group). In contrast, bNAbs plus any single latency-reversing agent did not show significant effects.

The second paper—published in PNAS by Tae-Wook Chun and colleagues—explores the effects of bNAb combinations on HIV isolated from the latently infected CD4 T cells of individuals on ART. To try and model the spread of HIV from the latent reservoir that occurs when ART is interrupted, laboratory experiments were conducted in which CD4 T cells sampled from uninfected individuals were exposed to HIV from the latent reservoir in the presence or absence of various bNAbs. Results showed that the most potent suppression of the reservoir-derived HIV was achieved by the bNAbs PGT121 (mean 2.4 log), VRC01 (2.1 log), and VRC03 (1.8 log). The authors also note: “viral isolates from 72%, 52%, and 44% of HIV-infected individuals we studied were neutralized (>2 log suppression) by PGT121, VRC01, and VRC03, respectively;” this finding underscores that not all circulating HIV is susceptible to every bNAb.

In discussing their results, the researchers suggest: “a combination of HIV-neutralizing monoclonal antibodies, particularly PGT121, VRC01, and VRC03, may provide sustained virologic remission in infected individuals following the discontinuation of ART.” Because bNAbs may have the potential to be administered infrequently, they argue this could represent an alternative to continuous ART. The paper concludes by recommending that, due to the variation in HIV susceptibility to bNAbs, “clinical trials involving passive immunization should include prescreening of HIV isolates from the persistent viral reservoirs of infected individuals with a panel of HIV-specific antibodies, to identify those that manifest the most potent suppressive activity against the patient viral isolates.”

Currently there are two ongoing phase I, first-in-human clinical trials of the monoclonal bNAbs 3BNC117 and VRC01. A study of PGT121 is also in the works. At the recent Forum for Collaborative HIV Research workshop in Washington DC, plans for a trial involving passive immunization with VRC01 in people with acute HIV infection were presented by Jintanat Ananworanich (the presentation is available on the Forum’s website). The administration of bNAbs in these studies is via injection, but there may be alternative possibilities. As covered previously on the blog, two research groups are testing adeno-associated virus (AAV) as a possible means to generate a permanent supply of circulating bNAbs after a single injection; although the original impetus for this work was the goal of preventing HIV acquisition, there is also interest in exploring the therapeutic potential. A phase I study of the approach in HIV-negative individuals is currently underway in the UK.

Latent reservoirs of HIV-1-infected cells are refractory to antiretroviral therapies (ART) and remain the major barrier to curing HIV-1. Because latently infected cells are long-lived, immunologically invisible, and may undergo homeostatic proliferation, a "shock and kill" approach has been proposed to eradicate this reservoir by combining ART with inducers of viral transcription. However, all attempts to alter the HIV-1 reservoir in vivo have failed to date. Using humanized mice, we show that broadly neutralizing antibodies (bNAbs) can interfere with establishment of a silent reservoir by Fc-FcR-mediated mechanisms. In established infection, bNAbs or bNAbs plus single inducers are ineffective in preventing viral rebound. However, bNAbs plus a combination of inducers that act by independent mechanisms synergize to decrease the reservoir as measured by viral rebound. Thus, combinations of inducers and bNAbs constitute a therapeutic strategy that impacts the establishment and maintenance of the HIV-1 reservoir in humanized mice.

PNAS Published online before print August 25, 2014, doi: 10.1073/pnas.1414148111

Several highly potent and broadly neutralizing monoclonal antibodies against HIV have recently been isolated from B cells of infected individuals. However, the effects of these antibodies on the persistent viral reservoirs in HIV-infected individuals receiving antiretroviral therapy (ART) are unknown. We show that several HIV-specific monoclonal antibodies—in particular, PGT121, VRC01, and VRC03—potently inhibited entry into CD4+ T cells of HIV isolated from the latent viral reservoir of infected individuals whose plasma viremia was well controlled by ART. In addition, we demonstrate that HIV replication in autologous CD4+ T cells derived from infected individuals receiving ART was profoundly suppressed by three aforementioned and other HIV-specific monoclonal antibodies. These findings have implications for passive immunotherapy as an approach toward controlling plasma viral rebound in patients whose ART is withdrawn.

A letter published yesterday in the New England Journal of Medicine describes the outcome of a recent attempt to repeat the HIV cure achieved in Timothy Brown. An HIV-positive individual requiring stem cell transplantation for the treatment of cancer (anaplastic large-cell lymphoma) was matched with a donor homozygous for the CCR5Δ32 mutation, which renders cells resistant to CCR5-tropic HIV. Pre-transplant analyses indicated that the majority of HIV in the individual was CCR5-tropic, but there was also evidence of HIV strains capable of entering cells via the CXCR4 (X4) receptor.

Antiretroviral therapy (ART) was interrupted during the transplantation procedure, but restarted afterward due to a viral load rebound to 93,390 copies. Analysis of the rebounding virus revealed the selection of mutations associated with X4-tropism, consistent with this virus gaining a selective advantage after the transplantation of cells resistant to CCR5-tropic HIV. Viral load was successfully re-suppressed by ART for nearly a year until the individual experienced a relapse of the lymphoma. The relapse necessitated a second ART interruption, leading to a viral load increase to 7,582,496 copies, and the individual died from the cancer shortly afterward. The researchers note the case illustrates that the presence of X4-tropic has the potential to undermine strategies that aim to cure HIV infection by knocking out the CCR5 receptor. The case report also underscores the importance of monitoring HIV tropism in other research studies looking to provide stem cells from CCR5Δ32 homozygous donors to HIV-positive people requiring transplants for the treatment of cancers. Two such studies are ongoing in the US: BMT CTN 0903 and IMPAACT P1107.

The question of why X4 virus did not emerge in Timothy Brown is addressed in an open access paper published in the August 15th issue of Clinical Infectious Diseases. Jori Symons and colleagues report that there was a minority population of HIV present prior to Brown’s transplant that showed evidence of X4 tropism (based on genetic analysis of the viral envelope). However, laboratory experiments revealed that these viruses required the presence of CCR5 in order to gain entry into cells, and could not replicate in cell lines lacking the receptor.

Several independent research groups have identified ingenol esters, compounds derived from the tropical shrub Euphorbia tirucalli, as potent activators of latent HIV. Papers published in the journals Virology and AIDS report that a specific semi-synthetic ingenol ester named 3-caproyl-ingenol (ING B) demonstrates the greatest promise and may represent a candidate for clinical trials. The compound appears to work by targeting the protein kinase C (PKC) pathway, similar to another known latency-reversing candidate prostratin but with less evidence of toxicity. At the International Workshop on HIV Persistence last December, Lucio Gama from Johns Hopkins University presented results from ING B studies in SIV-infected macaques indicating that the drug was safe to administer and showed activity against latent virus. Gama also offered a glimpse at preliminary data from the laboratory of Robert Siliciano suggesting that ING B potently activated latent HIV in CD4 T cells isolated from HIV-positive individuals on ART. Additional analyses are ongoing with a view to assessing if ING B can safely be advanced into human clinical trials, and whether additional modifications to the compound might enhance safety and activity.

The ability of HIV to establish long-lived latent infection is mainly due to transcriptional silencing of viral genome in resting memory T lymphocytes. Here, we show that new semi-synthetic ingenol esters reactivate latent HIV reservoirs. Amongst the tested compounds, 3-caproyl-ingenol (ING B) was more potent in reactivating latent HIV than known activators such as SAHA, ingenol 3,20-dibenzoate, TNF-α, PMA and HMBA. ING B activated PKC isoforms followed by NF-κB nuclear translocation. As virus reactivation is dependent on intact NF-κB binding sites in the LTR promoter region ING B, we have shown that. ING B was able to reactivate virus transcription in primary HIV-infected resting cells up to 12 fold and up to 25 fold in combination with SAHA. Additionally, ING B promoted up-regulation of P-TEFb subunits CDK9/Cyclin T1. The role of ING B on promoting both transcription initiation and elongation makes this compound a strong candidate for an anti-HIV latency drug combined with suppressive HAART.

HIV infection is not cleared by antiretroviral drugs due to the presence of latently infected cells that are not eliminated with current therapies and persist in the blood and organs of infected patients. New compounds to activate these latent reservoirs have been evaluated so that, along with HAART, they can be used to activate latent virus and eliminate the latently infected cells resulting in eradication of viral infection. Here we describe three novel diterpenes isolated from the sap of Euphorbia tirucalli, a tropical shrub. These molecules, identified as ingenols, were modified at carbon 3 and termed ingenol synthetic derivatives (ISD). They activated the HIV-LTR in reporter cell lines and human PBMCs with latent virus in concentrations as low as 10 nM. ISDs were also able to inhibit the replication of HIV-1 subtype B and C in MT-4 cells and human PBMCs at concentrations of EC50 0.02 and 0.09 µM respectively, which are comparable to the EC50 of some antiretroviral currently used in AIDS treatment. Control of viral replication may be caused by downregulation of surface CD4, CCR5 and CXCR4 observed after ISD treatment in vitro. These compounds appear to be less cytotoxic than other diterpenes such as PMA and prostratin, with effective dose versus toxic dose TI>400. Although the mechanisms of action of the three ISDs are primarily attributed to the PKC pathway, downregulation of surface receptors and stimulation of the viral LTR might be differentially modulated by different PKC isoforms.

OBJECTIVE: Although HAART effectively suppresses viral replication, it fails to eradicate latent viral reservoirs. The 'shock and kill' strategy involves the activation of HIV from latent reservoirs and targeting them for eradication. Our goal was to develop new approaches for activating HIV from latent reservoirs.

DESIGN: We investigated capacity of Ingenol B (IngB), a newly modified derivative of Ingenol ester that was originally isolated from a Brazilian plant in Amazon, for its capacity and mechanisms of HIV reactivation.

METHODS: Reactivation of HIV-1 by IngB was evaluated in J-Lat A1 cell culture model of HIV latency as well as in purified primary CD4 T cells from long-term HAART-treated virologically-suppressed HIV-infected individuals. The underlining molecular mechanisms of viral reactivation were investigated using flow cytometry, RT-qPCR and chromatin immunoprecipitation.

RESULTS: IngB is highly effective in reactivating HIV in J-Lat A1 cells with relatively low cellular toxicity. It is also able to reactivate latent HIV in purified CD4 T cells from HAART-treated HIV-positive individuals ex vivo. Our data show that IngB may reactivate HIV expression by both activating protein kinase Cδ-NF-κB pathway and directly inducing NF-κB protein expression. Importantly, IngB has a synergistic effect with JQ1 in latent HIV reactivation.

CONCLUSIONS: IngB is a new promising compound to activate latent HIV reservoirs. Out data suggest that formulating novel derivatives from Ingenol esters may be an innovative approach to develop new lead compounds to reactivate latent HIV.

The 20th International AIDS Conference (AIDS 2014) begins Sunday in Melbourne. A number of sessions and potentially interesting abstracts cover HIV cure research, and a two-day International AIDS Society (IAS) symposium on the topic immediately precedes the main meeting on July 19th and 20th (the program for the symposium is available online). Recommendations for some relevant sessions and abstracts are below, there is also an official IAS “Towards an HIV Cure” conference roadmap on the AIDS 2014 website.

Initial results from the Canadian pediatric HIV research mentioned in a recent blog post were published in Clinical Infectious Diseases on June 9th. The paper notes that Canada’s three pediatric HIV care institutions have a longstanding policy of administering triple drug antiretroviral therapy (ART) regimens to newborns considered at a high risk of HIV infection (either because the mother has detectable viral load at delivery or, in the absence of viral load results, if there is evidence of non-adherence to ART). Treatment is continued if HIV infection is subsequently confirmed. The researchers, inspired in part by the Mississippi baby case report (before the news of viral rebound emerged), searched their records for examples of children who had been started on ART within 72 hours of birth. A total of 136 were identified and, of those, 12 were confirmed to have acquired HIV infection. Four have since been maintained on ART with continuous viral load suppression; ages range from 2.5-7.5 years.

Consistent with other studies of early ART in infants, all four were found to lack detectable HIV-specific antibody and cellular immune responses, and displayed undetectable or extremely low levels of HIV DNA and RNA. In two of the children, tests for replication-competent virus were performed; in one case the result was negative and in the other 0.1 infectious units per million CD4 T cells were detected (the lowest level of detection possible with the assay). The researchers are scheduled to present an update on the cohort next week at the International AIDS Conference in Melbourne, during the “Challenges in Paediatrics” session on Tuesday July 22nd (abstract TUAB0206LB).

Regrettably, the notoriously unreliable media outlet The Daily Mail (often colloquially referred to as The Daily Fail) has already offered wildly misleading coverage of this research, with a headline on July 1st that read: “Three babies 'CURED' of HIV after being given revolutionary new vaccine.” As is typical, the exciting-sounding but mistaken article rapidly proliferated on the web, being copied on multiple other sites and social media. After complaints, the Mail made a small and completely inadequate correction to the headline, removing the imaginary vaccine and instead offering: “Three babies 'CURED' of HIV after being given revolutionary new medical treatment in the first hours of their lives” (the reference to a vaccine persists in the URL and page header). A few paragraphs down, the article flatly contradicts its own headline with an eminently sensible quote from Dr. Sharon Lewin: “At the moment, the doctors do not know whether they are in fact cured. The only way they can tell is if they stop the anti-HIV drugs and see if it comes back.” The importance of Lewin’s point has, sadly, since been further emphasized by the news about the viral load rebound in the child in Mississippi.

The Canadian researchers themselves, in a separate presentation at the 23rd Annual Canadian Conference on HIV/AIDS Research in May, documented an example of viral load rebound after ART interruption in another of the 12 children identified in their analysis (abstract appended below).

Although hopes of achieving a cure with early ART alone have now been diminished, studies like these remain essential for understanding the extent to which the HIV reservoir can potentially be reduced by early treatment, and for setting the stage for studies of interventions that might be able to target the residual amounts of virus for elimination.

BACKGROUND: An HIV-1 infected infant started on combination antiretroviral therapy (cART) at 30 hours of life was recently reported to have no detectable plasma viremia after discontinuing cART. The current study investigated the impact of early cART initiation on measures of HIV-1 reservoir size in HIV-1-infected children with sustained virologic suppression.

METHODS: Children born to HIV-1-infected mothers started on cART within 72 hours of birth at three Canadian centers were assessed. HIV serology, HIV-1-specific cell-mediated immune responses, plasma viremia, cell-associated HIV-1 DNA and RNA, presence of replication-competent HIV-1 and HLA genotype were determined for HIV-1-infected children with sustained virologic suppression.

RESULTS: Of 136 cART treated children, 12 were vertically infected (8.8%). In the four who achieved sustained virologic suppression HIV serology, HIV-1-specific cell-mediated immune responses (Gag, Nef) and ultrasensitive viral load were negative. HIV-1 DNA was not detected in enriched CD4+ T-cells of the four children (<2.6 copies/106 CD4+ T-cells), whereas HIV-1 RNA was detected (19.5-130 copies/1.5 µg RNA). No virion-associated HIV-1 RNA was detected following mitogenic stimulation of peripheral blood CD4+ T-cells (5.4-8.0 million CD4+ T-cells) in these four children, but replication competent virus was detected by quantitative co-culture involving a higher number of cells in one of two children tested (0.1 infectious units/106 CD4+ T-cells).

CONCLUSIONS: In perinatally HIV-1-infected newborns, initiation of cART within 72 hours of birth may significantly reduce the size of the HIV-1 reservoirs. Cessation of cART may be necessary to determine whether functional HIV cure can be achieved in such children.

BACKGROUND: The possibility of eradicating HIV following early initiation of combination antiretroviral therapy (cART) and sustained viral suppression (SVS) is of current interest. We report one such child who discontinued therapy and had rapid viral rebound.

RESULTS: An infant whose mother had inadequately controlled HIV infection at delivery, commenced nevirapine-based cART on the day of birth. HIV DNA PCR was positive on day 6 and initial viral load (VL) on day 28 was 808 c/mL. Maternal genotyping demonstrated NNRTI resistance, and cART was changed on day 25 to a lopinavir/ritonavir-based regimen. SVS was achieved at 175 days, and remained so for 2.5 years except for a single measurement of 40 c/mL. At age 3, HIV serology and ultrasensitive VL were negative. Cell-associated proviral DNA (detection limit 2.6 copies/μg DNA) was undetectable, whereas cell-associated RNA was 149 copies/1.5 μg RNA. Replication competent virus was not demonstrated in stimulated CD4+ T-cell co-culture (6.7 million cells). At age 3.25 years, cART was interrupted following adherence difficulties. Two and four weeks later, VL was 7797 c/mL and 11358 c/mL respectively. HIV-1 specific T-cell responses to gag and nef were also positive. Repeat serology is pending.

CONCLUSIONS: Viral resistance to the initial cART regimen, prolonged time to SVS and residual HIV replication (as evidenced by history of a single detectable VL) may have contributed to the development and persistence of viral reservoirs in this child. Caution should be exercised prior to attempting treatment interruption to evaluate for potential cure in this setting.

Understandably, there has been extensive media coverage of yesterday’s announcement that HIV has rebounded in the “Mississippi Baby” case. Although a full discussion of the implications will take time, there are some points that I think may be worth noting now:

The number of individuals considered cured of HIV infection has dwindled back to one: Timothy Ray Brown. It had been hoped that the child in Mississippi was another example, and for a brief time last year two adults from Boston were considered possibly cured. In all three of these cases, the evidence suggests that HIV reservoirs were reduced to extremely low levels but eventually a dormant, latently infected cell became activated and sparked a renewal of viral replication. Earlier this year it was reported that a second baby from Long Beach in California shows no detectable HIV after very early treatment, and some media outlets erroneously portrayed this case as potentially another example of a cure, but the infant remains on antiretroviral therapy. The sobering outcome in Mississippi further emphasizes that the absence of detectable HIV cannot be assumed to mean the virus has been cleared.

As highlighted by amfAR in their statement, the case underscores the challenges associated with attempting to measure the vanishingly small amounts of HIV that can persist, particularly in body tissues.

The fate of the clinical trial based on the Mississippi baby, IMPAACT P1115, may become a matter of controversy. A variety of opinions are reported in the current media coverage. Some scientists note that two years without the need for treatment is not trivial and represents a benchmark to try and build upon; based on this view, it is perhaps possible that the IMPAACT trial could attempt to establish how frequently such remissions occur, and whether they might last longer in some cases (close monitoring would certainly be required during interruptions to ensure treatment could be restarted as soon as any HIV rebound was detected). But other scientists argue that interrupting treatment would now be unethical (NPR quotes an ethicist making this argument). Further dialogue is clearly needed to reach agreement on how (or if) the trial should proceed.

Although yesterday’s news has dealt a severe blow to hopes that very early HIV treatment alone might be curative, the evidence remains clear that swift initiation of antiretroviral therapy after infection is associated with a significant reduction in the size of the HIV reservoir. For this reason, there is still broad consensus that early-treated individuals are ideal candidates for trials of interventions that aim to further reduce the reservoir or induce containment of any residual HIV. Such trials are already being planned in a cohort of early-treated adults in Thailand, using interventions such as therapeutic vaccination and infusions of broadly neutralizing antibodies (the design of the latter trial, RV397, was presented and discussed at the Regulatory Pathway for HIV Cure Research meeting).

The return of the Mississippi child to the media spotlight is also a reminder that the case arose from a bad situation, in that the mother had an undiagnosed infection and did not receive necessary prenatal healthcare. Ideally, all HIV-positive mothers should be able to access high quality, appropriate care to minimize the risk of perinatal transmission, and this remains a vital priority. Jim Merrell from the Prevention Justice Alliance wrote a commentary on this issue last year that is still relevant.

On June 17th, the Forum for Collaborative HIV Research convened a meeting in Washington DC to discuss regulatory issues relating to HIV cure research. Youtube videos of all the sessions, slide presentations and associated meeting materials (including an Excel file with detailed cure research trial information) are now available on the Forum’s HIV Cure Project webpage. A paper summarizing the outcomes from meeting will be forthcoming.

A news release issued today by the National Institute of Allergy and Infectious Diseases (NIAID) reports the disappointing news that viral load has rebounded in the child in Mississippi who had been considered possibly cured of HIV infection. The child is now nearly four years of age and HIV had remained undetectable without treatment for over two years, but recent routine laboratory testing revealed a viral load of 16,750 copies that was confirmed 72 hours later with a second measurement of 10,564 copies. HIV-specific antibodies are also now detectable and CD4 T cell numbers have declined. The child has been restarted on antiretroviral therapy and is said to be responding well. Genetic sequencing was used to establish that the rebounding HIV was derived from the virus present in the mother.

The news obviously has implications for the IMPAACT trial mentioned in my last blog post, but it is not yet clear what the effect on the trial will be; the NIAID release simply states: “the researchers planning the clinical trial will now need to take this new development into account.” In an interview with Forbes, Daniel Kuritzkes notes that the first goal of the trial is the initiation of early treatment in HIV-infected newborns and decisions about whether to interrupt ART will not be made until later.

The outcome in the Mississippi baby case suggests that early HIV treatment greatly reduced the number of latently infected cells, but some remained present and ultimately rekindled viral replication. There are parallels with the two adults known as the Boston patients, whose latent HIV reservoirs were significantly reduced by stem cell transplant procedures, leading to an extended delay in viral load rebound after stopping ART (but not a cure). The idea that reducing the number of latently infected cells can lead to a delay in reappearance of viral load after ART interruption has recently been proposed by Alison Hill and colleagues from the laboratory of Robert Siliciano. Hill’s mathematical modeling work was presented at CROI 2013 and has since been published in Quantitative Biology (see abstract and link to free full text PDF below). The results indicate that large reductions in the latent HIV reservoir can delay viral load rebound for many years or even decades. To completely prevent viral load rebound in most individuals, Hill estimates that a greater than six log (million-fold) diminution of the HIV reservoir would be required. The findings argue that in cases where HIV does not rebound after ART interruption, extremely long-term follow-up will be required in order to ascertain if a cure has been achieved.

Massive research efforts are now underway to develop a cure for HIV infection, allowing patients to discontinue lifelong combination antiretroviral therapy (ART). New latency-reversing agents (LRAs) may be able to purge the persistent reservoir of latent virus in resting memory CD4+ T cells, but the degree of reservoir reduction needed for cure remains unknown. Here we use a stochastic model of infection dynamics to estimate the efficacy of LRA needed to prevent viral rebound after ART interruption. We incorporate clinical data to estimate population-level parameter distributions and outcomes. Our findings suggest that approximately 2,000-fold reductions are required to permit a majority of patients to interrupt ART for one year without rebound and that rebound may occur suddenly after multiple years. Greater than 10,000-fold reductions may be required to prevent rebound altogether. Our results predict large variation in rebound times following LRA therapy, which will complicate clinical management. This model provides benchmarks for moving LRAs from the lab to the clinic and can aid in the design and interpretation of clinical trials. These results also apply to other interventions to reduce the latent reservoir and explain the observed return of viremia after months of apparent cure in recent bone marrow transplant recipients.

An open access paper published in the Journal of Infectious Diseases reports that earlier HIV suppression by ART is associated with significantly smaller HIV reservoirs in perinatally infected youths. The study, by Katherine Luzuriaga and colleagues, was initially presented at CROI last year and compared two groups of four individuals: one group started ART at 0.5–2.6 months of age and the other at 6-14.7 years of age. HIV DNA levels were significantly lower in the early-treated group, and replication-competent virus could only be detected in one out of the four, at a very low level, but was found in all of the late-treated study participants. Additionally, there was evidence of a decrease in HIV DNA levels over time in the early-treated group. At this year’s CROI, Deborah Persaud described a similar but much larger analysis that echoed these findings, and also identified negative or indeterminate HIV antibody tests as a marker for smaller reservoirs (the presentation can be viewed via webcast).

Both the Journal of Infectious Diseases paper and an accompanying commentary by Jonathan Li and Rajesh Gandhi note that the results are consistent with prior studies in adults demonstrating that early initiation of ART is associated with a significantly lower HIV reservoir (see abstracts and links to free full text for two examples below). One implication is that some early-treated individuals may ultimately be able to safely interrupt ART, but the authors emphasize that this needs to be tested in carefully conducted clinical trials. Several cautionary examples of HIV rebound despite undetectable or extremely low reservoirs prior to ART interruption are cited, including the Boston patients and a case report from the laboratory of Tae-Wook Chun.

An additional example involving a 3-year-old child started on ART within 72 hours after birth was reported recently at the Canadian Association for HIV Research conference. According to an article in the Globe & Mail, an ART interruption was prompted by adherence difficulties and viral load rebounded from undetectable levels to 7,797 copies in two weeks and 11,358 copies in four (although the article uses the word “skyrocketed,” these viral load levels are actually low for untreated pediatric HIV infection and the apparent increase from the second to fourth week is small and within the variability of the assay). ART has since been restarted and viral load once again suppressed. The researchers presenting this case noted some differences with the Mississippi baby, including a longer time to viral load suppression (six months versus 19 days) and an instance of a borderline detectable viral load prior to the ART interruption.

A clinical trial investigating immediate ART in babies born to HIV-positive mothers who did not receive prevention of mother-to-child transmission (PMTCT) is about to be launched by the International Maternal, Pediatric, Adolescent AIDS Clinical Trials (IMPAACT) Network. An entry for the trial is now posted in the clinicaltrials.gov database and additional information, including the protocol, is available on the IMPAACT website. The primary aim is to assess whether the apparent remission of HIV infection that has been achieved in the Mississippi baby can be duplicated in other infants.

Results. Plasma viremia was not detected in any ET youth but was detected in all LT youth (median 8 copies/ml; p=0.03). PBMC proviral load was significantly lower in ET youth (median 7 copies per million peripheral blood mononuclear cells) than in LT youth (median 181 copies; p=0.03). Replication competent virus was recovered from all LT youth but only one ET youth. Decay in proviral DNA was noted in all four ET youth in association with limited T cell activation and absent to minimal HIV-1 specific immune responses.

Conclusions. Early effective cART from infancy significantly limits circulating levels of proviral and replication-competent HIV-1 and promotes continuous decay of viral reservoirs. Continued cART with reduction in HIV-1 reservoirs over time may facilitate HIV-1 eradication strategies.

Methods. From a cohort of patients with early HIV infection (<6 months duration since infection) we identified persons who started ART early (<6 months after infection) or later (≥2 years after infection) and maintained ≥2 years of virologic suppression; at-risk HIV-negative persons were controls. We measured CD4(+)/CD8(+) T-cell activation (percent CD38(+)/HLA-DR(+)) and HIV reservoir size (based on HIV DNA and cell-associated RNA levels).

METHODS: This was a prospective, single-centre cohort study of HIV-1-infected patients on effective cART. Total cell-associated HIV DNA levels and T cell counts before and during treatment were used to identify factors predictive of OVIR status {i.e. low HIV DNA level [<2.3 log10 copies/10(6) peripheral blood mononuclear cells (PBMCs)], together with normalization of the absolute/relative CD4+ T cell counts and CD4+/CD8+ ratio}.

RESULTS: A total of 307 patients were enrolled, of whom 35 started cART during PHI (<4 months post-infection) and 272 during CHI. HIV DNA decay was modelled with a non-linear mixed-effects model that showed two phases of HIV DNA decay, both of which were significantly more pronounced in the PHI group. At the end of follow-up, after a median of 4 years of viral suppression (<50 copies/mL), HIV DNA levels were lower in the PHI group than in the CHI group (median = 2.15 versus 2.84 log10 copies/10(6) PBMCs; P< 0.0001). Immune reconstitution was more rapid and sustained in the PHI group (median = 883 versus 619 CD4+ cells/mm(3); 41% versus 31% CD4+; CD4+/CD8+ 1.31 versus 0.77; all P< 0.0001). Finally, OVIR status was obtained in 19/35 (54%) and 7/272 (3%) patients in the PHI and CHI groups (P< 0.0001), respectively. In a logistic regression analysis, cART initiation during PHI (OR = 16, 95% CI = 3.5-72.3) and HIV DNA level <3.3 log10 before treatment (OR = 4.8, 95% CI = 1.2-19.3) were independently predictive of OVIR status.